Subcultured cells are frequently used in biological experiments to understand the nature of a particular substance such as a protein or a chemical. When a substance is tested on cells that are subcultured in wells of a multi-well plate, it is important to obtain reproducible and repeatable test results to be able to confidently rely on those results. In the past, methods of distributing cells for biological experiments provided random and unequal volumes of cells in each well of a multi-well plate that was used for testing. The unequal volume of cells created large variations in results when an equal amount of a substance was tested on the cells in each well. Therefore, scientists had to frequently perform several tests to obtain a result that was duplicated a sufficient number of times to be considered reliable.
Distributing equal volumes of attached confluent living cells in each well of a multi-well plate, including 4, 8, 12, 24, 48, 96, 384 or more well plates, has been a long-standing problem in high throughput screening bioassays partly because the trypsinization process does not guarantee equal volumes of cells in each small confluent cell group. During the trypsinization process, the enzyme trypsin reacts to the surface of attached confluent cells and gradually separates a cell or cells on the edge or surface of very large confluent cell groups. As a result, the cell suspension consists of varying sizes of confluent cell groups including single cells, small cell clusters, large cell clusters and huge cell clumps. Raising the amount of trypsin or prolonging the treatment of trypsin does not cure the separation problem because cells in the middle of a confluent cell group may never be trypsinized at all and an over-treatment with the trypsin enzyme may kill the cells that are separated early in the trypsinization process.
Adding to the difficulty in distributing an equal volume of cells in each well of a multi-well plate is the non-uniformly distributed groups of cells in the cell suspension. As such, distributing an equal volume of the cell suspension in each well of a multi-well plate does not guarantee that equal volumes of cells are present in each volume of the cell suspension. Although very large confluent cell groups are sometimes artificially picked out to avoid clogging pipette tips, the distribution of a non-uniform cell suspension to each well of a multi-well plate unavoidably and directly causes a large variation in the volume of cells in each well of a multi-well plate. Because past attached cell-based biological testing resulted in a large standard deviation, the reliability, repeatability and reproducibility of the results were questionable. Accordingly, there remains a need to obtain a uniform distribution of cells in a cell suspension as well as a need to distribute equal volumes of attached confluent living cells in individual wells of a multi-well plate to reduce the standard deviation and to improve the reproducibility, repeatability and reliability of biological test results.